γ-Secretase: Successive Tripeptide and Tetrapeptide Release from the Transmembrane Domain of β-Carboxyl Terminal Fragment

<jats:p>Amyloid β protein (Aβ), a pathogenic molecule associated with Alzheimer's disease, is produced by γ-secretase, which cleaves the β-carboxyl terminal fragment (βCTF) of β-amyloid precursor protein in the middle of its transmembrane domain. How the cleavage proceeds within the membrane has long been enigmatic. We hypothesized previously that βCTF is cleaved first at the membrane–cytoplasm boundary, producing two long Aβs, Aβ<jats:sub>48</jats:sub>and Aβ<jats:sub>49</jats:sub>, which are processed further by releasing three residues at each step to produce Aβ<jats:sub>42</jats:sub>and Aβ<jats:sub>40</jats:sub>, respectively. To test this hypothesis, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) to quantify the specific tripeptides that are postulated to be released. Using CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxyl-1-propanesulfonate)-reconstituted γ-secretase system, we confirmed that Aβ<jats:sub>49</jats:sub>is converted to Aβ<jats:sub>43/40</jats:sub>by successively releasing two or three tripeptides and that Aβ<jats:sub>48</jats:sub>is converted to Aβ<jats:sub>42/38</jats:sub>by successively releasing two tripeptides or these plus an additional tetrapeptide. Most unexpectedly, LC-MS/MS quantification revealed an induction period, 3–4 min, in the generation of peptides. When extrapolated, each time line for each tripeptide appears to intercept the same point on the<jats:italic>x</jats:italic>-axis. According to numerical simulation based on the successive reaction kinetics, the induction period exists. These results strongly suggest that Aβ is generated through the stepwise processing of βCTF by γ-secretase.</jats:p>